Abstract
BackgroundWe reported previously that amoeboid microglial cells in the postnatal rat brain expressed 2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNPase) both in vivo and in vitro; however, the functional role of CNPase in microglia has remained uncertain. This study extended the investigation to determine CNPase expression in activated microglia derived from cell culture and animal models of brain injury with the objective to clarify its putative functions.MethodsThree-day-old Wistar rats were given an intraperitoneal injection of lipopolysaccharide to induce microglial activation, and the rats were killed at different time points. Along with this, primary cultured microglial cells were subjected to lipopolysaccharide treatment, and expression of CNPase was analyzed by real-time reverse transcription PCR and immunofluorescence. Additionally, siRNA transfection was employed to downregulate CNPase in BV-2 cells. Following this, inducible nitric oxide synthase, IL-1β and TNF-α were determined at mRNA and protein levels. Reactive oxygen species and nitric oxide were also assessed by flow cytometry and colorimetric assay, respectively. In parallel to this, CNPase expression in activated microglia was also investigated in adult rats subjected to fluid percussion injury as well as middle cerebral artery occlusion.ResultsIn vivo, CNPase immunofluorescence in activated microglia was markedly enhanced after lipopolysaccharide treatment. A similar feature was observed in the rat brain after fluid percussion injury and middle cerebral artery occlusion. In vitro, CNPase protein and mRNA expression was increased in primary microglia with lipopolysaccharide stimulation. Remarkably, inducible nitric oxide synthase, IL-1β, TNF-α, reactive oxygen species and nitric oxide were significantly upregulated in activated BV-2 cells with CNPase knockdown. siRNA knockdown of CNPase increased microglia migration; on the other hand, microglial cells appeared to be arrested at G1 phase.ConclusionsThe present results have provided the first morphological and molecular evidence that CNPase expression is increased in activated microglia. CNPase knockdown resulted in increased expression of various inflammatory mediators. It is concluded that CNPase may play an important role as a putative anti-inflammatory gene both in normal and injured brain.Electronic supplementary materialThe online version of this article (doi:10.1186/s12974-014-0148-9) contains supplementary material, which is available to authorized users.
Highlights
We reported previously that amoeboid microglial cells in the postnatal rat brain expressed 2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNPase) both in vivo and in vitro; the functional role of CNPase in microglia has remained uncertain
CNPase expression is increased in microglia after lipopolysaccharide treatment in postnatal rats To investigate the function of CNPase in microglial cells in the developing brain with LPS stimulation, we profiled the expression of CNPase in activated microglia in the brain, as well as primary cultured microglial cells subjected to LPS treatment
(See figure on previous page.) Figure 5 CNPase knockdown increased inducible nitric oxide synthesis, interleukin-1 beta (IL-1β) and tumor necrosis factor-alpha (TNF-α) expression induced by lipopolysaccharide in BV-2 cells. (A) Reverse transcription polymerase chain reaction analysis of inducible nitric oxide synthase (iNOS), IL-1β and TNF-α gene expression in BV-2 cells transfected with control small interfering RNA, transfected with control siRNA + lipopolysaccharide (LPS), and transfected with CNPase siRNA and CNPase siRNA + LPS
Summary
Three-day-old Wistar rats were given an intraperitoneal injection of lipopolysaccharide to induce microglial activation, and the rats were killed at different time points. Primary cultured microglial cells were subjected to lipopolysaccharide treatment, and expression of CNPase was analyzed by real-time reverse transcription PCR and immunofluorescence. CNPase expression in activated microglia was investigated in adult rats subjected to fluid percussion injury as well as middle cerebral artery occlusion. Injection of lipopolysaccharide To investigate CNPase expression in activated microglia in vivo, 3-day-old postnatal rats were weighed and given a single intraperitoneal injection of LPS (1 mg/kg of body weight, diluted in 250 μl saline; Sigma-Aldrich, St Louis, MO, USA; Cat. No L2654). The pups were sacrificed at 1, 3, and 6 hours after LPS injection (n = 3 rats at each time point interval for each group).
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